Treatment Of Normal Human Keratinocytes Research Articles

P37 Drug repurposing for treatment of recessive dystrophic epidermolysis bullosa cutaneous squamous cell carcinoma

Abstract Background Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited skin disorder that leads to severe wounding and blistering of the skin. Patients with RDEB develop aggressive cutaneous squamous cell carcinoma (cSCC) with a mortality rate of approximately 90% by age 55 years. The current primary treatment is wide excision of tumours, often leading to amputations. Identifying new therapeutic avenues for RDEB cSCC remains a priority. Here, we approach this in two ways. Firstly, we identified a SMAD4-dependent sphingosine pathway response to endogenous transforming growth factor (TGF)-β, essential for RDEB cSCC proliferation which could be targeted as a potential therapeutic strategy. Secondly, we utilized a US Food and Drug Administration (FDA) screen of > 3000 drugs to identify potential druggable pathways that could be used to treat RDEB cSCC. Methods RDEB cSCC cell lines were used to test Siponimod, a sphingosine-1 receptor modulator, on proliferation, clonogenicity and invasion in vitro. Siponimod was used in vivo to test tumour growth effect of targeting the sphingosine pathway. We carried out an FDA-approved drug screen on four RDEB cSCC cell lines using the Opera Phenix high-throughput microscope to identify targets. Results Treatment of RDEB cSCC cell lines with Siponimod showed compromised proliferation compared with untreated cells in addition to significant reduction in colony formation. There was a significant decrease in the invasive capability of RDEB SCC cells. Treatment of normal human keratinocytes showed no effect on proliferation. Our FDA-approved screen identified multiple drugs that target MEK, mechanistic target of rapamycin complex, histone deacetylase, signal transducer and activator of transcription, and topoisomerase I/II in the top 50 hits. The most common target was DNA damage/repair pathways, but cytoskeletal signalling, transmembrane transporters and proteases were identified as other potential avenues for treatment of RDEB cSCC. Conclusions There is an unmet need for therapeutic options in RDEB cSCC. Here, we have demonstrated the potential of repurposing previously licensed drugs to identify treatment avenues for RDEB cSCC.

S100A7 (psoriasin) inhibits human epidermal differentiation by enhanced IL-6 secretion through IκB/NF-κB signalling.

Psoriasin (S100A7), a member of the S100 protein family, is a well-known antimicrobial peptide and a signalling molecule which regulates cellular function and is highly expressed in hyperproliferative skin conditions such as atopic dermatitis (AD) and psoriasis with disrupted skin barrier function. However, its role in epidermal differentiation remains unknown. We examined the effect of S100A7 on epidermal differentiation in normal human keratinocytes (NHKs) and on a reconstituted human epidermis model. When NHKs were exposed to disruptive stimuli such as Staphylococcus aureus, ultraviolet irradiation and retinoic acid, the secretion of S100A7 into the culture medium increased and the expression of epidermal differentiation markers decreased. Treatment of NHKs with S100A7 significantly inhibited epidermal differentiation by reducing the expression of keratin 1, keratin 10, involucrin and loricrin and by increasing the expression of abnormal differentiation markers (keratin 6 and keratin 16). We verified that the MyD88-IκB/NF-κB signal cascade was activated via RAGE after S100A7 treatment, resulting in the upregulation of interleukin-6. Finally, we confirmed that S100A7 is a negative regulator of epidermal differentiation using a reconstituted human epidermis model. This study suggests that S100A7-related signalling molecules could be potent targets for recovering skin barrier function in AD and psoriasis where S100A7 is accumulated excessively.

Staphylococcus aureus inhibits terminal differentiation of normal human keratinocytes by stimulating interleukin-6 secretion

BackgroundStaphylococcus aureus (S. aureus) is found on the skin of approximately 90% of patients with atopic dermatitis and approximately 20% of apparently healthy subjects. S. aureus induces keratinocytes and immune cells to secrete immunoregulatory factors that cause epidermal barrier dysfunction in atopic skin. ObjectiveThis study examined factors that cause epidermal permeability barrier dysfunction in skin colonized by S. aureus. MethodsWe examined the effect of S. aureus on keratinocyte differentiation in the stratum corneum (SC) of in vivo skin, normal human keratinocytes (NHKs) and a reconstructed human epidermis (RHE) model. The fold change in expression of the terminal differentiation markers and the level of secreted cytokines were investigated. ResultsThe SC displayed decreased expression of keratin 10 (KRT 10). NHKs treated with S. aureus extracts increased expression of interleukin (IL)-6 and significantly reduced expression of the terminal differentiation markers KRT 1, KRT 10, loricrin (LOR), and filaggrin (FLG); however, the expression of basal layer markers (KRT 5, KRT 14) remained unchanged. Treatment of NHKs with an anti-IL-6 antibody in combination with IL-6 or the S. aureus extracts inhibited the decrease in KRT 10 mRNA or protein expression. After the RHEs were exposed to the S. aureus extracts, KRT 1 and KRT 10 protein levels decreased. ConclusionsThese findings suggest that S. aureus inhibits the terminal differentiation of keratinocytes by stimulating IL-6 secretion.

Normal Human Keratinocytes Pretreated with Damaged mRNA Are Resistant to UVB Damage and Exhibit Increased DNA Repair

Treatment of normal human keratinocytes with UVC-irradiated rabbit globin mRNA 24 h before and after UVB exposure increased the survival of the human keratinocytes. We also observed that UVC-damaged mRNA reduced the formation of sunburn cells in skin models. We next tested the effects of UVC-damaged mRNA on cellular repair of DNA. DNA repair was evaluated using 2 assay methods. The first method used a damaged plasmid that is transfected back into the cell where it is repaired by the host cell repair mechanism. In these experiments, we observed that externally added UVC-damaged rabbit globin mRNA enhanced the repair of a plasmid transfected into the host keratinocyte cells. The second method used to determine the effects on DNA repair was direct immunostaining for thymidine-thymidine dimers (TT dimers) in histological sections of the skin models. Skin models were irradiated with UVB and then fixed immediately or after 24 h and stained for TT dimers. UVB irradiation immediately caused an increase in the number of stained keratinocytes in the skin. The number of stained cells decreased in skin fixed 24 h after UVB. This is due to repair of the TT dimers and their removal. Sections of skin models pretreated with UV-damaged mRNA exhibit greater removal of these TT dimers after 24 h. The above evidence suggests that damaged mRNA can trigger a host cell DNA repair pathway.

Differential Responses of S100A2 to Oxidative Stress and Increased Intracellular Calcium in Normal, Immortalized, and Malignant Human Keratinocytes

S100A2 is a calmodulin-like, p53-inducible, homodimeric protein that is readily oxidized in keratinocytes subjected to oxidative stress. Here we compare the redox status and subcellular distribution of S100A2 in normal human keratinocytes, immortalized keratinocytes (HaCaT), and malignant keratinocytes (A431) as a function of oxidative stress and intracellular Ca2+ levels. Normal human keratinocytes displayed strong nuclear and moderate cytoplasmic S100A2 immunoreactivity. HaCaT and A431 cells, which lack normal p53, expressed S100A2 in similar patterns but in 4- to 8-fold lower amounts. H2O2 treatment of normal human keratinocytes caused a reduction of nuclear S100A2 staining accompanied by an increase in cytoplasmic S100A2 staining, with a delayed time course (0.5-1 h) relative to S100A2 oxidative crosslinking (15 min). This phenomenon, consistent with translocation of S100A2 from the nucleus to the cytoplasm, could also be induced in normal human keratinocytes by increasing intracellular Ca2+ levels with the ionophore A23187. Sulfhydryl reducing agents blocked these changes, whether induced by H2O2 or increased intracellular Ca2+ levels. A temporal correlation was identified between S100A2 translocation at 1 h and loss of cell viability at 24 h after H2O2 treatment. A431 and HaCaT cells were strongly resistant to H2O2-induced S100A2 crosslinking, S100A2 translocation, and cell death. Increased intracellular Ca2+ levels caused prominent translocation of S100A2 in normal human keratinocytes and HaCaT, but not in A431 cells. These results identify S100A2 oxidation and translocation as markers for early cellular responses to oxidative stress, which are markedly attenuated in immortalized and malignant keratinocytes.

Treatment of Normal Human Keratinocytes with 2,3,7,8-Tetrachlorodibenzo-p-dioxin Causes a Reduction in Cell Number, but No Increase in Apoptosis

We have examined the effect of TCDD on the growth of normal human keratinocytes. TCDD is a ubiquitous environmental toxicant that causes a severe dermatopathology in humans, which is known as chloracne. The cell biological basis of this pathology remains unknown. We conducted growth experiments in preconfluent normal human keratinocytes with both low (0.05 mM) and standard (0.66 mM) extracellular calcium concentrations in the media. TCDD treatment reduced the number of adherent keratinocytes relative to controls in media containing 0.05 or 0.66 mM calcium. Based on these observations, we speculated that the decrease in the cell number of TCDD-treated cultures might be the result of increased apoptosis. Analysis of nucleosomal fragmentation, nuclear morphology, and caspase-3 activity in keratinocytes reveals no increase in the characteristics of apoptosis in response to TCDD treatment. We therefore conclude that TCDD impacts on keratinocyte homeostasis independent of apoptosis.

Cholesterol Sulfate Activates Transcription of Transglutaminase 1 Gene in Normal Human Keratinocytes

Cholesterol sulfate and transglutaminase 1 are essential for the process of keratinization. Cholesterol sulfate is formed during keratinization and activates the eta isoform of protein kinase C. Transglutaminase 1 is a key enzyme for formation of the cornified envelope in terminally differentiated keratinocytes. In this study, we demonstrated that cholesterol sulfate acts as a transcriptional activator of the transglutaminase 1 gene in normal human keratinocytes. Growth of normal human keratinocytes was inhibited by cholesterol sulfate, but not by its parental cholesterol. Treatment of normal human keratinocytes with cholesterol sulfate induced activity of transglutaminase 1 in a dose- and time-dependent manner. Activation of transcription of transglutaminase 1 by cholesterol sulfate was demonstrated by northern blotting analysis, whereas that by cholesterol was not. In order to identify a cholesterol sulfate responsive region in the transglutaminase 1 gene, plasmids were constructed containing a luciferase reporter gene ligated to deletion fragments of the 5' upstream region of the tranglutaminase 1 gene and were transfected into normal human keratinocytes. Transfected cells were treated with cholesterol sulfate, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and a high concentration of Ca2+. Our results indicate that the responsive element(s) for cholesterol sulfate and phorbol ester is located upstream of the human transglutaminase 1 gene at a position(s) between -819 and -549, whereas the responsive element for Ca2+ is located at a position between -79 and -49.

A new function of calphobindin I (annexin V)

Calphobindin I (CPB-I, annexin V) is an anticoagulant protein purified from human placenta; it is a member of the annexin family that binds phospholipids in a calcium-dependent manner. In this study, we discovered and examined a new function of CPB-I: promotion of both the migration and the urokinase-type plasminogen activator (uPA) activity of normal human keratinocytes (NHK). While the treatment of NHK with a 10-micrograms/ml concentration of CPB-I for 24 h or 48 h caused an approximate 30% increase in the migration of NHK (compared with the no treatment), migration was inhibited when anti-CPB-I monoclonal antibodies (i.e. A46 and A180) were added along with the CPB-I. Moreover, while the treatment of NHK with a CPB-I concentration greater than 10 micrograms/ml caused a significant increase in the activity of secreted uPA, reflected in an approximately 40% increase in cell migration, uPA activity was inhibited both by cycloheximide and by monoclonal antibodies. This significant increase of secreted uPA was seen 8 h after the addition of CPB-I. Specific binding of CPB-I to NHK had a Kd value of 95.2 nM (equivalent to a CPB-I had no effect on NHK proliferation. Furthermore, CPB-I enhanced reepithelialization when it was applied locally twice a day to full-thickness cutaneous wounds made in male rats. Our results show that, during an injury, CPB-I helps reepithelialization through the promotion of both uPA synthesis and migration of keratinocytes without stimulating their proliferation.

Effect of Natural Retinoids on the Lipogenesis of Cultured Human Keratinocytes

Retinoids are known to be potent modulators of keratinocyte differentiation. In this report, the effect of all-trans-retinoic acid (TRA),13-cis-retinoic acid (CRA), retinol (ROL) and all-transretinal (RAL) on the lipogenesis of normal human keratinocytes (NHK) cultured on plastic was investigated. Measurement of 14C-acetate incorporation after one week treatment of NHK with retinoids, showed that all four compounds inhibited total lipogenesis in a concentration-dependent fashion. TRA and RAL exhibited comparable activity, as determined by their concentration inhibiting 50% of the lipogenesis (IC50) of 4.0 × 10-9M and 2.5 × 10-9M, respectively, being the most potent lipogenesis inhibitors, whereas ROL and CRA exhibited IC50 of 4.5 × 10-8M and 8.0 × 10-8M, respectively. However, retinoid-treated (e.g. TRA and CRA) NHK did not exhibit lipid patterns significantly different from that of vehicle treated cells, as determined by thin layer chromatography.